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Post-transcriptional modifications of RNA strongly influence RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before mass-spectrometry identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA-MS increases both coverage and throughput, while the EMLC LC-MS orthogonally separates RNA of different length and charge. The combination of the two methods offers a broadly applicable platform to improve length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.

Histone variants are a universal means to alter the biochemical properties of nucleosomes, implementing local changes in chromatin structure. H2A.Z, one of the most conserved histone variants, is incorporated into chromatin by SWR1-type nucleosome remodelers. Here, we summarize recent advances toward understanding the transcription-regulatory roles of H2A.Z and of the remodeling enzymes that govern its dynamic chromatin incorporation. Tight transcriptional control guaranteed by H2A.Z nucleosomes depends on the context provided by other histone variants or chromatin modifications, such as histone acetylation. The functional cooperation of SWR1-type remodelers with NuA4 histone acetyltransferase complexes, a recurring theme during evolution, is structurally implemented by species-specific strategies.

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, degradation, as well as human disease states such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to firstly efficiently derivatize the ribonucleosides and quantitatively analyze them by a liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate That this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlighted the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust, and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.

Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed low glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited markedly reduced GR protein levels. De novo or pre-existing mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.

Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In Drosophila, the single DOMINO chromatin regulator complex is thought to combine both activities via an unknown mechanism. Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. Drosophila generates the same diversity by alternative splicing of a single gene., eLife digest Cells contain a large number of proteins that control the activity of genes in response to various signals and changes in their environment. Often these proteins work together in groups called complexes. In the fruit fly Drosophila melanogaster, one of these complexes is called DOMINO. The DOMINO complex alters gene activity by interacting with other proteins called histones which influence how the genes are packaged and accessed within the cell. DOMINO works in two separate ways. First, it can replace certain histones with other variants that regulate genes differently. Second, it can modify histones by adding a chemical marker to them, which alters how they interact with genes. It was not clear how DOMINO can do both of these things and how that is controlled; but it was known that cells can make two different forms of the central component of the complex, called DOM-A and DOM-B, which are both encoded by the same gene. Scacchetti et al. have now studied fruit flies to understand the activities of these forms. This revealed that they do have different roles and that gene activity in cells changes if either one is lost. The two forms operate as part complexes with different compositions and only DOM-A includes the TIP60 enzyme that is needed to modify histones. As such, it seems that DOM-B primarily replaces histones with variant forms, while DOM-A modifies existing histones. This means that each form has a unique role associated with each of the two known behaviors of this complex. The presence of two different DOMINO complexes is common to flies and, probably, other insects. Yet, in other living things, such as mammals and yeast, their two roles are carried out by protein complexes originating from two distinct genes. This illustrates a concept called convergent evolution, where different organisms find different solutions for the same problem. As such, these findings provide an insight into the challenges encountered through evolution and the diverse solutions that have developed. They will also help us to understand the ways in which protein activities can adapt to different needs over evolutionary time.

Chromatin accessibility complex/ATP-utilizing chromatin assembly and remodeling factor help to establish basal transcriptional repression, conceivably through improving the regular spacing of nucleosomes in euchromatin., The chromatin remodeling complexes chromatin accessibility complex and ATP-utilizing chromatin assembly and remodeling factor (ACF) combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression remains unclear. Here, we explored the influence of Drosophila melanogaster chromatin accessibility complex/ACF on transcription by using complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of an Acf knock-out allele showed that only lowly expressed genes are derepressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together, our results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, with T lineage ALL (T-ALL) accounting for approximately 20% of cases. Many T-ALL patients present with high risk clinical features, and receive aggressive multi-agent genotoxic therapies that pose a substantial risk of long-term adverse health effects. Glucocorticoids (GCs) are an integral part of current treatment strategies for T-ALL and other lymphoid cancers. Despite decades of clinical use, the molecular mechanisms underlying GC efficacy and resistance are incompletely understood. Limited initial response to GCs correlates with poor outcome, and secondary GC resistance is frequently observed in relapsed patients. Thus, identifying drugs that either enhance initial responses or prevent resistance to GCs could significantly improve the treatment of T-ALL and other lymphoid malignancies. Our laboratory performed retroviral insertional mutagenesis (RIM) in wild-type (KrasWT) and KrasG12D "knock-in" mutant mice to generate a panel of primary transplantable T-ALLs that recapitulate the genetic heterogeneity found in human cancers. We used these reagents to assess the efficacy of treatment with the GC dexamethasone (DEX) alone and in combination with the PI3 kinase (PI3K) inhibitor GDC-0941. We observed a robust and significant overall response to 15 mg/kg/day DEX in cohorts of recipient mice transplanted with 10 independent parental KrasWT (n=5) and KrasG12D (n=5) T-ALLs, which was modestly enhanced by combined treatment with 125 mg/kg/day GDC-0941. Prolonged in vivo treatment resulted in outgrowth of 65 independent relapsed T-ALLs, many of which harbor novel retroviral integrations. We verified intrinsic drug resistance in a number of these relapsed leukemias by transplanting them into secondary recipients and treating these mice with 15 mg/kg/day DEX. Intriguingly, 23 (35%) of the T-ALLs that relapsed after an initial response to treatment exhibited markedly reduced GC receptor (GR) protein expression, suggesting a novel and common mechanism for evading GC-induced cell death. Similarly, analysis of T-ALL patient samples showed that low GR expression is rare in diagnostic specimens, but enriched in relapsed/refractory cases. We have identified two likely mechanisms of GR down-regulation in relapsed mouse T-ALLs including a nonsense mutation in the gene encoding GR and a retroviral integration in a putative distal GR promoter region that greatly reduces mRNA expression. We also performed transcriptome (RNA-seq) analysis in one sensitive parental and corresponding resistant KrasWT T-ALL after short-term in vivo DEX treatment, and observed a dramatic reduction in the number of differentially expressed genes in the resistant versus parental leukemia. We recently generated transcriptomes from this panel of 10 primary RIM-induced T-ALLs with the goal of identifying a GC response signature, and also to assess the ability of resistant cells that have lost or retained GR expression to activate this program. Finally, in contrast to previous studies in this genetically accurate in vivo preclinical model (Dail et al. Nature 2014), very few relapsed T-ALLs showed reduced Notch intracellular domain (NICD) expression relative to the corresponding parental leukemia suggesting that loss of Notch1 activation is not a major mechanism contributing to DEX resistance. Taken together, these data reveal an unexpected and common putative mechanism of GC resistance in T-ALL that can inform the development of treatment strategies for relapsed/refractory patients. We are currently focusing on analyzing transcriptome data and performing whole exome sequencing in order to uncover additional mechanisms of resistance to DEX, and then using these data to identify and investigate targeted inhibitors that might overcome GC resistance in vivo. Disclosures Dail: Genentech, Inc.: Employment. Sampath:Genentech: Employment.